Modulators of regulatory proteins

ABSTRACT

A modulator of regulatory cellular events occurring intracellularly which are mediated by regulatory proteins containing a ‘death domain’ motif is provided. The ‘death domain’ is a regulatory portion of the regulatory proteins, and the modulator is capable of interacting with one or more ‘death domain’ motifs contained in the regulatory proteins and affecting the regulatory action of one or more of the regulatory proteins. The modulator preferably is capable of interacting with ‘death domain’ motifs within p55-TNF-R, FAS/APO1-R, NGF-R, MORT-1, RIP, TRADD, or ankyrin. A method for producing the modulators is also provided. The modulators are useful for modulating functions mediated in cells by proteins containing the ‘death domain’.

FIELD OF THE INVENTION

The present invention is generally in the field of regulatory proteinswhich exert their effects by intracellular signaling processes which aremediated by regulatory elements (domains or motifs) contained within theintracellular domains of these proteins. More specifically, the presentinvention concerns new modulators being, proteins, peptides, antibodiesor analogs or fragments of any thereof, and organic compounds which arecapable of interacting with, or binding to, the newly discovered ‘deathdomain’ motif present in a wide range of related and unrelated proteins,for example, receptors of the TNF/NGF family such as p55 TNF-R, FAS-R,NGF-R, a related protein MORT-1, proteins known as TRADD and REP and theunrelated protein ankyrin 1. These new modulators are capable ofmodulating or regulating the activity of the proteins which contain the‘death domain’ motif.

BACKGROUND OF THE INVENTION AND PRIOR ART

There is a very large group of regulatory proteins which exert theirregulatory effects on cells by way of intracellular signaling processes,mediated by regulatory portions or motifs contained within theseproteins. Members of this group of proteins include, receptors belongingto the TNF/NGF family of receptors, such as, for example, the p55 andp75 TNF receptors (p55 and p75 TNF-Rs), the NGF receptor (NGF-R) and theFas/APO1 protein (also called the FAS-ligand receptor or FAS-R, andhereinafter will be called FAS-R); these receptors being characterizedby having an extracellular ligand-binding domain, a transmembrane domainand an intracellular (IC) domain, which intracellular domain, orportions thereof, is involved in the mediation of the intracellularsignaling events initiated by the binding of the ligand to theextracellular domain. Other members of this group include variousintracellular proteins, for example, the cytoskeleton-associatedstructural proteins, the ankyrins, which have a regulatory domain thatis possibly involved in the ability of these proteins to associate withor bind to other cytoskeletal proteins, e.g., spectrin, or to othertransmembrane proteins. Yet another member of this group is the recentlyidentified MORT-1 protein (also called HF1, see copending IL 112002 andIL 112692), which is capable of binding specifically to theintracellular domain of the FAS-P, and which is also capable ofself-association and of mediating, in a ligand-independent manner,cytotoxic effects on cells. In MORT-1, a regulatory domain was alsoidentified (see IL 112692).

Tumor Necrosis Factor (TNF-α) and Lymphotoxin (TNF-β) (hereinafter, TNFrefers to both TNF-α-and TNF-β) are multifunctional pro-inflammatorycytokines formed mainly by mononuclear phagocytes, which have manyeffects on cells (Wallach, 1986; and Beutler et al, 1987). Both TNF-αand TNF-β initiate their effects by binding to specific cell surfacereceptors. Some of the effects are likely to be beneficial to theorganism: they may destroy, for example tumor cells or virus infectedcells and augment antibacterial activities of granulocytes. In this way,TNF contributes to the defense of the organism against tumors andinfectious agents and contributes to the recovery from injury. Thus, TNFcan be used as an anti-tumor agent in which application it binds to itsreceptors on the surface of tumor cells and thereby initiates the eventsleading to the death of the tumor cells. TNF can also be used as ananti-infectious agent.

However, both TNF-α and TNF-β also have deleterious effects. There isevidence that over-production of TNF-α can play a major pathogenic rolein several diseases. Thus, effects of TNF-α, primarily on thevasculature, are now known to be a major cause for symptoms of septicshock (Tracey et al, 1986). In some diseases, TNF may cause excessiveloss of weight (cachexia) by suppressing activities of adipocytes and bycausing anorexia, and TNF-α was thus called cachectin. It was alsodescribed as a mediator of the damage to tissues in rheumatic diseases(Beutler et al, 1987) and as a major mediator of the damage observed ingraft-versus-host reactions (Piguet et al, 1987). In addition, TNF isknown to be involved in the process of inflammation and in many otherdiseases.

Two distinct, independently expressed, receptors, the p55 and p75TNF-Rs, which bind both TNF-α and TNF-β specifically, initiate and/ormediate the above noted biological effects of TNF. These two receptorshave structurally dissimilar intracellular domains suggesting that theysignal differently (see Hohmann et al, 1989; Engelmann et al, 1990;Brockhaus et al, 1990; Loetscher et al, 1990; Schall et al, 1990; Nopharet al, 1990; Smith et al, 1990; and Heller et al, 1990). However, thecellular mechanisms, for example, the various proteins and possiblyother factors, which are involved in the intracellular signaling of thep55 an p75 TNF-Rs have yet to be elucidated. (In IL 109632 there aredescribed for the first time, new proteins capable of binding to theintracellular domains of p55 and p75 TNF-Rs, these intracellular domainsbeing called, respectively, p75 IC and p55 IC.) It is this intracellularsignaling, which occurs usually after the binding of the ligand, i.e.,TNF (α or β), to the receptor, that is responsible for the commencementof the cascade of reactions that ultimately result in the observedresponse of the cell to TNF.

As regards the above mentioned cytocidal effect of TNF, in most cellsstudied so far, this effect is triggered mainly by the p55 TNF-R.Antibodies against the extracellular domain (ligand binding domain) ofthe p55 TNF-R can themselves trigger the cytocidal effect (see EP412486) which correlates with the effectively of receptor cross-linkingby the antibodies, believed to be the first step in the generation ofthe intracellular signaling process. Further, mutational studies(Brakebusch et al, 1992; Tartaglia et al, 1993) have shown that thebiological function of the p55 TNF-R depends on the integrity of itsintracellular domain, and accordingly it has been suggested that theinitiation of intracellular signaling leading to the cytocidal effect ofTNF occurs as a consequence of the association of two or moreintracellular domains of the p55 TNF-R. Moreover, TNF (α and β) occursas a homotrimer and as such has been suggested to induce intracellularsignaling via the p55 TNF-R by way of its ability to bind to and tocross-link the receptor molecules, i.e., cause receptor aggregation. Inco-pending IL 109632 and IL 111125, there is described how the p55 ICand p55 DD can self-associate and induce, in a ligand-independentfashion, TNF-associated effects in cells.

Another member of the TNF/NGF superfamily of receptors is the FASreceptor (FAS-R) which has also been called the Fas antigen, acell-surface protein expressed in various tissues and sharing homologywith a number of cell-surface receptors including TNF-R and NGF-R. TheFAS-R mediates cell death in the form of apoptosis (Itoh et al, 1991),and appears to serve as a negative selector of autoreactive T cells,i.e., during maturation of T cells, FAS-R mediates the apoptotic deathof T cells recognizing self-antigens. It has also been found thatmutations in the FAS-R gene (lpr) cause a lymphoproliferation disorderin mice that resembles the human autoimmune disease systemic lupuserythematosus (SLE) (Watanabe-Fukunaga et al, 1992). The ligand for theFAS-R appears to be a cell-surface associated molecule carried by,amongst others, killer T cells (or cytotoxic T lymphocytes—CTLs), andhence when such CTLs contact cells carrying FAS-R, they are capable ofinducing apoptotic cell death of the FAS-R-carrying cells. Further, amonoclonal antibody has been prepared that is specific for FAS-R, thismonoclonal antibody being capable of inducing apoptotic cell death incells carrying FAS-R, including mouse cells transformed by cDNA encodinghuman FAS-R (Itoh et al, 1991).

It has also been found that various other normal cells, besides Tlymphocytes, express the FAS-R on their surface and can be killed by thetriggering of this receptor. Uncontrolled induction of such a killingprocess is suspected to contribute to tissue damage in certain diseases,for example, the destruction of liver cells in acute hepatitis.Accordingly, finding ways to restrain the cytotoxic activity of FAS-Rmay have therapeutic potential.

Conversely, since it has also been found that certain malignant cellsand HIV-infected cells carry the FAS-R on their surface, antibodiesagainst FAS-R, or the FAS-R ligand, may be used to trigger the FAS-Rmediated cytotoxic effects in these and thereby provide a means forcombating such malignant cells or HIV-infected cells (see Itoh et al,1991). Finding yet other ways for enhancing the c-cytotoxic activity ofFAS-R may therefore also have therapeutic potential.

In co-pending IL 109632, IL 111125 and IL 112002 there is described thatthe intracellular domain of FAS-R, the so-called FAS-IC, is capable ofself-association and contains within this intracellular domain a regioncalled the ‘death domain’ (DD) which is primarily responsible for theself-association of the FAS-IC. This ‘death domain’ shares sequencehomology with the p55 TNF-R, ‘death domain’ (p55 DD).

It has been a long felt need to provide a way for modulating thecellular response to TNF (α or β) and FAS-R ligand, for example, inpathological situations as mentioned above, where TNF or FAS-R ligand isover-expressed it is desirable to inhibit the TNF- or FAS-Rligand-induced cytocidal effects, while in other situations, e.g., woundhealing applications, it is desirable to enhance the TNF effect, or inthe case of FAS-R, in tumor cells or HIV-infected cells it is desirableto enhance the FAS-R mediated effect.

A number of approaches have been made by the present inventors (see forexample, European Application Nos. EP 186833, EP 308378, EP 398327 andEP 412486) to regulate the deleterious effects of TNF by inhibiting thebinding of TNF to its receptors using anti-TNF antibodies or by usingsoluble TNF receptors (being essentially the soluble extracellulardomains of the receptors) to compete with the binding of TNF to the cellsurface-bound TNF-Rs. Further, on the basis that TNF-binding to itsreceptors is required for the TNF-Induced cellular effects, approachesby the present inventors (see for example IL 101769 and itscorresponding EP 568925) have been made to modulate the TNF effect bymodulating the activity of the TNF-Rs. Briefly, EP 568925 (IL 101769)relates to a method of modulating signal transduction and/or cleavage inTNF-Rs whereby peptides or other molecules may interact either with thereceptor itself or with effector proteins interacting with the receptor,thus modulating the normal functioning of the TNF-Rs. In EP 568925 thereis described the construction and characterization of various mutant p55TNF-Rs, having mutations in the extracellular, transmembranal, andintracellular domains of the p55 TNF-R. In this way regions within theabove domains of the p55 TNF-R were identified as being essential to thefunctioning of the receptor, i.e., the binding of the ligand (TNF) andthe subsequent signal transduction and intracellular signaling whichultimately results in the observed TNF-effect on the cells. Further,there is also described a number of approaches to isolate and identifyproteins, peptides or other factors which are capable of binding to thevarious regions in the above domains of the TNF-R, which proteins,peptides and other factors may be involved in regulating or modulatingthe activity of the TNF-R. A number of approaches for isolating andcloning the DNA sequences encoding such proteins and peptides; forconstructing expression vectors for the production of these proteins andpeptides; and for the preparation of antibodies or fragments thereofwhich interact with the TNF-R or with the above proteins and peptidesthat bind various regions of the TNF-F, are also set forth in EP 568925.However, no description is made in EP 568925 of the actual proteins andpeptides which bind to the intracellular domains of the TNF-Rs (e.g.,p55 TNF-R), nor is any description made of the yeast two-hybrid approachto isolate and identify such proteins or peptides which bind to theintracellular domains of TNF-Rs. Similarly, heretofore there has been nodisclosure of proteins or peptides capable of binding the intracellulardomain of FAS-R.

Thus, when it is desired to inhibit the effect of TNF, or the FAS-Rligand, it would be desirable to decrease the amount or the activity ofTNF-Rs or FAS-R at the cell surface, while an increase in the amount orthe activity of TNF-Rs or FAS-R would be desired when an enhanced TNF orFAS-R ligand effect is sought. To this end the promoters of both the p55TNF-R and the p75 TNF-R have been sequenced, analyzed and a number ofkey sequence motifs have been found that are specific to varioustranscription regulating factors, and as such the expression of theseTNF-Rs can be controlled at their promoter level, i.e., inhibition oftranscription from the promoters for a decrease in the number ofreceptors, and an enhancement of transcription from the promoters for anincrease in the number of receptors (see IL 104355 and IL 109633).Corresponding studies concerning the control of FAS-R at the level ofthe promoter of the FAS-R gene have yet to be reported.

Further, it should also be mentioned that, while it is known that thetumor necrosis factor (TNF) receptors, and the structurally-relatedreceptor FAS-F, trigger in cells, upon stimulation by leukocyte-producedligands, destructive activities that lead to their own demise, themechanisms of this triggering are still little understood. Mutationalstudies indicate that in FAS-R and the p55 TNF receptor (p55-R)signaling for cytotoxicity involve distinct regions within theirintracellular domains (Brakebusch et al, 1992; Tartaglia et al, 1993;Itoh et al, 1993). These regions (the ‘death domains’) have sequencesimilarity. The ‘death domains’ of both FAS-R and p55-R tend toself-associate. Their self-association apparently promotes that receptoraggregation which is necessary for initiation of signaling (see IL109632, IL 111125 and IL 112002, as well as Song et al, 1994; Wallach etal, 1994; Boldin et al, 1995) and at high levels of receptor expressioncan result in triggering of ligand-independent signaling (IL 109632, IL111125 and Boldin et al, 1995).

The ankyrins constitute a family of proteins that control interactionsbetween integral membrane components and cytoskeletal elements and arefound in a wide range of tissues such as brain tissue and inerythrocytes, the erythrocyte ankyrin being the best characterized. Theankyrins are intracellular proteins associated with the cytoskeletalelements of the cell and have three domains: an upper domain involved inbinding to the intracellular domains of transmembrane proteins, thisupper domain containing the well-known repeats, the so-called ankyrinrepeats; a middle domain which is involved in binding to spectrin, i.e.,the binding of spectrin to transmembrane proteins via the ankyrins; anda C-terminal or lower (or third) domain, which is the regulatory domainthat is capable of being phosphorylated, this domain regulating theactivity of the other two domains when phosphorylated ordephosphorylated. This latter regulatory domain also has three parts: amiddle part that can be deleted by alternative splicing naturally, andhence some ankyrins have this part and others do not; and two otherparts, less well characterized (for a review on the ankyrins, see Lux etal, 1990 and Lambert et al, 1993).

It should be noted however, as is set forth hereinbelow, that inaccordance with the present invention, it has been discovered that theupper part of the above noted regulatory (C-terminal) domain of ankyrincontains a so-called ‘death domain’ motif, which may function to mediatethe binding of proteins together (activity of the first two ankyrindomains), or may function conformationally to regulate the ankyrinprotein.

The NGF-R is a low affinity NGF receptor which is not wellcharacterized. The NGF-R is considered to be involved in growthregulation, such as its possible involvement in signalingintracellularly for NGF-induced effects. However, a recent publicationdiscloses that overexpression of NGF-R in the absence of NGF can causecell death. Thus, NGF-R appears to have a regulatory role in cellviability (see Rabizadeh et al 1993).

It should be noted however, as is set forth hereinbelow, that inaccordance with the present invention, it has been discovered that theNGF-R contains a ‘death domain’ motif in its intracellular domain, whichmay be involved in the mediation of the intracellular events associatedwith the regulatory role played by NGF-R with regards to cell viability.

MORT-1 is a recently discovered protein that binds to the intracellulardomain of FAS-R, is capable of self-association and can activate cellcytotoxicity on its own. Hence, MORT-1 is also a regulatory proteininvolved in intracellular signaling processes. It was also discoveredthat MORT-1 has a “death domain” motif that is associated with itsobserved biological activity (see co-pending IL 112002 and IL 112692).

Two further intracellular proteins, RIP (Stanger et al, 1995) and TRADD(Hsu et al, 1995), that bind to the intracellular domains of p55 TNF-Ror FAS-R and apparently take part in the induction of their cytocidaleffect, have recently been cloned. All three proteins, MORT-1, RIP andTRADD, were found to contain the sequence motif shared between the‘death domains’ of the intracellular domains of p55-TNF-R and FAS-R. Asin the receptors, the ‘death domain’ motifs (DD) in the threeintracellular proteins seem to be sites of protein-protein interaction.The three proteins interact with the p55-TNF-R and FAS-R intracellulardomains by the binding of their DDs to those in the receptors, and inboth TRADD and RIP (though not in MORT-1) the DDs self-associate. It hasnow been found that MORT-1 and TRADD bind differentially to FAS-R andp55 TNF-R and also bind to each other. Moreover, both bind effectivelyto RIP.

Interference of the interaction between the above three intracellularproteins will result in modulation of the effects caused by thisinteraction. Thus, inhibition of TRADD binding to MORT-1 may modulateFAS-R—p55 TNF-R interaction. Inhibition of RIP in addition to the aboveinhibition of TRADD binding to MORT-1 may further modulate FAS-R—p55TNF-R interaction.

Monoclonal antibodies raised against the ‘death domain’ of the p55TNF-R, specifically against the binding site or sites of TRADD and RIPcan also be used to inhibit or prevent binding of these proteins andthus cause modulation of the interaction between the FAS-R and the p55TNF-R.

In a way analogous to that noted above in respect of TNF/TNF-R andFAS-ligand/FAS-R, there is also a need to provide a way for modulatingthe activity of the above noted proteins, i.e., ankyrin, NGF-R andMORT-1, namely, to inhibit their activity when it is associated withdetrimental effects, e.g., disease/disorder-related cell cytotoxicity orconformational changes in cell-shape; or to enhance their activity whenthis is desired, e.g., for directed destruction of diseased cells, etc.

In the co-pending applications, IL 109632, IL 111125, IL 112002 and IL112692, there are described proteins which are involved in themodulation of the activity of receptors belonging to the TNF/NGFreceptor family, these proteins being characterized by being capable ofbinding/associating with the intracellular domains of one or more ofthese receptors.

The present invention concerns modulators, such as proteins, peptides,antibodies and organic compounds, which are capable ofinteracting/binding with one or more so-called ‘death domain’ motifs inthe intracellular domains of proteins containing such motifs, theseproteins being related, e.g., members of the TNF/NGF receptor family orproteins related thereto, e.g., MORT-1, or unrelated proteins, e.g.,ankyrins. These modulators are characterized by recognizing generalstructural features common to the ‘death domain’ motifs of the ‘deathdomain’ motif-containing proteins, and by also recognizing specificstructural features present in each of the different ‘death domain’motifs of these proteins.

Accordingly, it is one aim of the invention to provide modulators, asnoted above, capable of binding to or interacting with the ‘deathdomain’ motifs of one or more of the ‘death domain’ motif-containingproteins and thereby modulating the activity of these proteins.

Another aim of the invention is to provide antagonists (e.g.,antibodies) to one class of these modulators, namely thenaturally-occurring proteins or peptides which bind to ‘death domain’motif-containing proteins, and which antagonists may be used to inhibitthe signaling process, when desired, when such ‘death domain’motif-binding proteins or peptides are positive signal effectors (i.e.,induce signaling), or to enhance the signaling process, when desired,when such ‘death domain’ motif-binding proteins are negative signaleffectors (i.e., inhibit signaling).

Yet another aim of the invention is to use such ‘death domain’motif-binding proteins or peptides to isolate and characterizeadditional proteins or factors, which may, for example, be involvedfurther downstream in the signaling process, and/or to isolate andidentify other receptors further upstream in the signaling process towhich these ‘death domain’ motif-binding proteins bind, and hence, inwhose function they are also involved.

Moreover, it is an aim of the present invention to use theabove-mentioned ‘death domain’ motif-binding proteins as antigens forthe preparation of polyclonal and/or monoclonal antibodies thereto. Theantibodies, in turn, may be used for the purification of the new ‘deathdomain’ motif-binding proteins from different sources, such as cellextracts or transformed cell lines.

Furthermore, these antibodies may be used for diagnostic purposes, e.g.,for identifying disorders related to abnormal functioning of cellulareffects mediated by the various proteins belonging to the group of‘death domain’ motif-containing proteins.

A further aim of the invention is to provide pharmaceutical compositionscomprising the above ‘death domain’ motif-binding modulators (proteins,peptides, organic molecules), and pharmaceutical compositions comprisingthe ‘death domain’ motif-binding protein or peptide antagonists, for thetreatment or prophylaxis of conditions related to the activity of the‘death domain’ motif-containing proteins, for example, such compositionscan be used to enhance the TNF or FAS ligand effect or effects mediatedby NGF-R, MORT-1, RIP, TRADD and ankyrin, or to inhibit the TNF or FASligand effect or effects mediated by depending on the above noted natureof the ‘death domain’ motif-binding modulators or antagonists thereofcontained in the composition.

A still further aim of the invention is to use the various ‘deathdomain’ motifs of the proteins containing them for the design andsynthesis of complementary peptides and organic molecules which will bemodulators of these proteins.

SUMMARY OF THE INVENTION

The present invention is based on the surprising and unexpected findingthat there exists a so-called ‘death domain’ motif in a wide range ofproteins some of which are related and others which are not related. Forexample, this ‘death domain’ motif has been found in p55 TNF-R, FAS-R,NGF-R, MORT-1, RIP and TRADD which are related to each other, as well asin the unrelated protein, ankyrin 1.

As noted above, the ‘death domain’ motif of the proteins containing thismotif is located in the intracellular regulatory domain of theseproteins. Hence, the ‘death domain’ motif appears to be involved in aregulatory function associated with cell viability (cell death) as wellas cell shape/conformation, this function being effected at (i.e., inthe case of receptors containing this motif) or close to (i.e., in thecase of structural intracellular proteins, e.g., ankyrin) the cellsurface. Moreover, the observation, in accordance with the presentinvention, that the ‘death domain’ motif is conserved amongst a widerange of related and non-related proteins indicates that this motif mayhave an important regulatory function.

Accordingly, the present invention provides a modulator of regulatorycellular events occurring intracellularly that are mediated byregulatory proteins containing a ‘death domain’ motif which is aregulatory portion of said proteins, said modulator being capable ofinteracting with one or more of the ‘death domain’ motifs contained insaid regulatory proteins and affecting the regulatory action of one ormore of said regulatory proteins.

In particular, the present invention provides:

(i) a modulator selected from the group comprising naturally-derived‘death domain’ motif-binding proteins and peptides and analogs andderivatives thereof capable of interacting with one or more of said‘death domain’ motifs;

(ii) a modulator selected from the group of synthetically producedcomplementary peptides, synthesized by using as substrates the ‘deathdomain’ motif sequences of said regulatory proteins containing ‘deathdomain’ motifs, said complementary peptides being capable of interactingwith one or more of said ‘death domain’ motifs;

(iii) a modulator selected from the group comprising antibodies oractive fragments thereof capable of interacting with one or more of said‘death domain’ motifs;

(iv) a modulator selected from the group of organic compounds capable ofinteracting with one or more of said ‘death domain’ motifs, said organiccompounds being derived from known compounds and selected by using said‘death domain’ motifs as a substrate in a binding assay, or beingsynthesized using said ‘death domain’ motifs as a substrate fordesigning and synthesizing said organic compounds;

(v) a modulator selected from the group of peptides or polypeptidesderived from naturally occurring ‘death domain’ motif sequences, saidpeptides or polypeptides being capable of interacting with one or moreof said ‘death domain’ motifs, and analogs and derivatives of saidpeptides or polypeptides capable of interacting with one or more of said‘death domain’ motifs;

(vi) a modulator of any one of (i)-(v) wherein said modulator is furthercharacterized by being capable of recognizing the general ‘death domain’motif sequence features common to the ‘death domain’ motifs of ‘deathdomain’ motif containing proteins, and being capable of recognizing oneor more of the specific ‘death domain’ motifs of said proteins, saidspecific sequence features being specific to each ‘death domain’ motifsequence of each of said proteins;

(vii) a modulator of any one of (i)-(vi) wherein said modulator iscapable of interacting with one or more of the ‘death domain’ motifscontained within the proteins belonging to the group comprising p55TNF-R, FAS-R, NGF-R, MORT-1, REP, TRADD and ankyrin 1;

(viii) a modulator of (vii) wherein said modulator is furthercharacterized by being capable of interacting with common sequencefeatures of the ‘death domain’ motifs of said group of proteins, saidcommon sequence features comprising the group of common amino acidresidues W (tryptophan), L (leucine), I (isoleucine), A (alanine), D(aspartic acid), E (glutamic acid), T (threonine), R (arginine) and Y(tyrosine) at the location within said ‘death domain’ motifs shown inFIG. 1.

The present invention also provides a DNA sequence encoding a modulatorbeing a protein, peptide or polypeptide or an analog of any one of (1),(ii) and (vii).

An embodiment of the DNA sequence of the invention is a DNA sequenceencoding a naturally derived protein or peptide selected from the groupconsisting of:

(a) a cDNA sequence derived from the coding region of a native ‘deathdomain’ motif-binding protein or peptide, (b) DNA sequences capable ofhybridization to a sequence of (a) under moderately stringent conditionsand which encode a biologically active ‘death domain’ motif-bindingprotein or peptide; and

(c) DNA sequences which are degenerate as a result of the genetic codeto the DNA sequences defined in (a) and (b) and which encode abiologically active ‘death domain’ motif-binding protein or peptide.

Other embodiments of the DNA sequence of the invention are:

(i) DNA sequence encoding a ‘death domain’ motif-binding protein orpeptide capable of binding to the ‘death domain’ motif of one or more ofthe proteins of the group comprising p55 TNF-R, FAS-R, NGF-R, MORT-1 andankyrin 1.

(ii) DNA sequence encoding a peptide or polypeptide derived from thenaturally-occurring ‘death domain’ motif sequence of the ‘death domain’motif-containing proteins.

(iii) DNA sequence encoding a peptide or polypeptide derived from the‘death domain’ motif sequence of any one of the proteins of the groupcomprising p55 TNF-R, FAS-R, NGF-R, MORT-1, RIP, TRADD and ankyrin 1.

Furthermore, there is also provided (a) a protein, peptide orpolypeptide and analogs of any one thereof encoded by a DNA sequence ofthe invention said protein, peptide, polypeptide and analogs beingcapable of binding to or interacting with one or more of the ‘deathdomain’ motifs of one or more ‘death domain’ motif containing, proteins;

(b) a vector comprising a DNA sequence of the invention;

(c) a vector of (b) capable of being expressed in a eukaryotic hostcell;

(d) a vector of (b) capable of being expressed in a prokaryotic hostcell;

(e) transformed eukaryotic or prokaryotic, host cells containing avector of (b), (c) or (d);

(f) a method for producing the protein, peptide, polypeptide or analogsof (a) comprising growing the transformed host cells of (e) underconditions suitable for the expression of said protein, peptide,polypeptide or analogs, effecting post-translational modifications ofsaid protein, peptide, polypeptide or analogs as necessary for obtentionthereof and extracting said expressed protein, peptide, polypeptide oranalogs from the culture medium of said transformed cells or from cellextracts of said transformed cells;

(g) antibodies or active fragments or derivatives thereof, specific forthe protein, peptide, polypeptide or analogs of (a).

The present invention also provides a method for the modulation of theTNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, orthe functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1or by other proteins containing a ‘death domain’ motif comprisingtreating said cells with one or more proteins, peptides, polypeptides oranalogs selected from the group consisting of the proteins, peptides,polypeptides or analogs of the invention (see (a) above), all beingcapable of binding to or interacting with the ‘death domain’ motif andmodulating the activity of said ‘death domain’ motif-containingproteins, wherein said treating of said cells comprises introducing intosaid cells said one or more proteins, peptides, polypeptides or analogsin a form suitable for intracellular introduction thereof, orintroducing into said cells a DNA sequence encoding said one or moreproteins, peptides, polypeptides or analogs in the form of a suitablevector carrying said sequence, said vector being capable of effectingthe insertion of said sequence into said cells in a way that saidsequence is expressed in said, cells.

An embodiment of the above method is a method wherein said treating ofsaid cells is by transfection of said cells with a recombinant animalvirus vector comprising the steps of:

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein (ligand) that is capable of binding toa specific cell surface receptor on the surface of said cell to betreated and a second sequence encoding a protein selected from theproteins, peptides, polypeptides and analogs of the invention, saidprotein, peptide, polypeptide or analogs, when expressed in said cellsbeing capable of modulating the activity of said ‘death domain’motif-containing protein; and

(b) infecting said cells with said vector of (a).

Another method of the invention is a method for modulating the TNF orFAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or thefunctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 orby other proteins containing a ‘death domain’ motif, comprising treatingsaid cells with antibodies or active fragments or derivatives thereof,of the invention (see (g) above), said treating being by application ofa suitable composition containing said antibodies, active fragments orderivatives thereof to said cells, said composition being formulated forintracellular application.

Yet another method of the invention is a method for modulating the TNFor FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or thefunctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 orby other proteins containing a ‘death domain’ motif, comprising treatingsaid cells with an oligonucleotide sequence selected from a sequenceencoding an antisense sequence of at least part of the sequence of theinvention as noted above, said oligonucleotide sequence being capable ofblocking the expression of at least one of the ‘death domain’motif-binding proteins or peptides.

An embodiment of the above method is a method wherein saidoligonucleotide sequence is introduced to said cells via a virus vectoras noted above wherein said second sequence of said virus encodes saidoligonucleotide sequence.

Other methods of the invention are:

(i) A method for treating tumor cells ‘or HIV-infected cells or otherdiseased cells, comprising:

-   -   (a) constructing a recombinant animal virus vector carrying a        sequence encoding a viral surface protein that is capable of        binding to a specific tumor cell surface receptor or        HIV-infected cell surface receptor or receptor carried by other        diseased cells and a sequence encoding a protein selected from        the proteins, peptides, polypeptides and analo2s of the        invention, said protein, peptide, polypeptide or analogs when        expressed in said tumor, IEV-infected, or other diseased cell        being capable of killing said cell; and    -   (b) infecting said tumor or HIV-infected cells or other diseased        cells with said vector of (a).

(ii) A method for modulating the TNF or FAS-R ligand effect on cellsmediated by p55 TNF-R and FAS-R, or the functions mediated in cells byNGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a‘death domain’ motif, comprising applying the ribozyme procedure inwhich a vector encoding a ribozyme sequence capable of interacting witha cellular mRNA sequence encoding a protein or peptide of the invention,is introduced into said cells in a form that permits expression of saidribozyme sequence in said cells, and wherein when said ribozyme sequenceis expressed in said cells it interacts with said cellular mRNA sequenceand cleaves said mRNA sequence resulting in the inhibition of expressionof said protein or peptide in said cells.

(iii) A method for isolating and identifying proteins, peptides, factorsor receptors capable of binding to the ‘death domain’ motif-bindingproteins or peptides of the invention, comprising applying the procedureof affinity chromatography in which said protein or peptide of theinvention is attached to the affinity chromatography matrix, saidattached protein is brought into contact with a cell extract andproteins, factors or receptors from cell extract which bound to saidattached protein are then eluted, isolated analyzed.

(iv) A method for isolating and identifying proteins, capable of bindingto the ‘death domain’ motif-binding proteins or peptides of theinvention, comprising applying the yeast two-hybrid procedure in which asequence encoding said ‘death domain’ motif-binding protein is carriedby one hybrid vector and sequence from a cDNA or genomic DNA library arecarried by the second hybrid vector, the vectors then being used totransform yeast host cells and the positive transformed cells beingisolated, followed by extraction of the said second hybrid vector toobtain a sequence encoding a protein which binds to said ‘death domain’motif-binding protein.

The present invention also provides a pharmaceutical composition for themodulation of the TNF- or FAS-R ligand-effect on cells mediated by p55TNF-R and FAS-R, or the functions mediated in cells by NGF-R, MORT-1,REP, TRADD, ankyrin 1 or by other proteins containing a ‘death domain’motif comprising, as active, ingredient a modulator of the invention.

Embodiments of the pharmaceutical compositions of the invention include:

(i) a pharmaceutical composition for modulating the TNF- or FAS-Rligand-effect on cells mediated by p55 TNF-R and FAS-R, or the functionsmediated in cells by NGF-R, MORT-1, REP, TRADD, ankyrin 1 or by otherproteins containing a ‘death domain’ motif, comprising, as activeingredient, a recombinant animal virus vector encoding a protein capableof binding a cell surface receptor and encoding a protein or peptide oranalogs thereof of the invention.

(ii) a pharmaceutical composition for modulating the TNF or FAS-R ligandeffect on cells mediated by p55 TNF-R and FAS-R, or the functionsmediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by otherproteins containing a ‘death domain’ motif, comprising as activeingredient, an oligonucleotide sequence encoding an anti-sense sequenceof the sequence of the invention.

A still further method of the invention is a method for isolating andidentifying a protein capable of binding to the ‘death domain’ motifs of‘death domain’ motif-containing proteins comprising applying theprocedure of non-stringent southern hybridization followed by PCRcloning, in which a sequence or parts thereof of the invention is usedas a probe to bind sequences from a cDNA or genomic DNA library, havingat least partial homology thereto, said bound sequences then amplifiedand cloned by the PCR procedure to yield clones encoding proteins havingat least partial; homology to said sequences of the invention.

In addition, the present invention also provides a method for designingdrugs that are capable of modulating the activity of ‘death domain’motif-containing proteins, comprising the procedures described herein inExamples 3 and 4.

Other aspects and embodiments of the present invention are also providedas arising from the following detailed description of the invention.

It should be noted that, where used throughout, the following terms“Modulation/Mediation of the TNF or FAS-R ligand effect on cellsmediated by p55 TNF-R and FAS-R, or the functions mediated in cells byNGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a‘death domain’ motif”’ are understood to encompass in vitro as well asin vivo treatment.

Moreover, where used throughout, the antibodies of the invention and themethods using these antibodies, include so-called “humanized” antibodiesor the use thereof

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically the sequence homology of the ‘death domain’motif in MORT-1 (SEQ ID NO:5), p55 TNF-R (SEQ ID NO:3), Fas/APO1 (FAS-R)(SEQ ID NO:1), low affinity NGF receptor (NGF-R) (SEQ ID NO:4), and theC-terminal part of the regulatory domain in ankyrin 1 (Ankyrin 1) (SEQID NO:2), as described in Example 1.

FIG. 2 depicts interactions of the ‘death domains’ of the P55-R,Fas/APOI, MORT-1, TRADD and. RIP in a yeast two-hybrid test, and theeffect of lbr^(cg)-like mutations in these proteins on theirinteractions. Assessment of the interaction of Gal4 hybrid constructsencompassing the following human proteins, truncated upstream to theirDD motifs: p55-R (residues 326-426), FAS-R (residues 210-319), MORT-1(residues 92-208), TRADD (residues 195-312) and RIP (residues 261-372),as well as of the following points mutants of these proteins: p55-RL35IN, FAS-R V238N, MORT-1 V121N, and RIP F308N, whose mutation siteswithin the DDs correspond to that found in the FAS-R of the lpr^(cg)mice. Each cDNA insert was introduced both into the Gal4 DNA bindingdomain (DBD) and the Gal4 activation domain (AD) constructs (pGBT9 abdpGAD-GH), and the binding of the inserts in both constructs to all otherinserts within transfected SFY526 yeasts was assessed by aβ-galactosidase expression filter assay. The results are presented interms of the time required for development of strong color. ND—not done.

FIG. 3 is a diagrammatic illustration of the DD interactions observed inthe yeast two-hybrid tests. The lengths and thicknesses of the arroseconnecting the DD icons correspond to the intensity of the interactions,as observed in the experiment described in FIG. 2.

FIG. 4 depicts interactions of MORT-1, TRADD and RIP within transfectedHeLa cells. MORT-1 (nucleotides 19-753 in GenBank accession numberU24231), fused at is N-terminus with the FLAG octapeptides, and the DDsof TRADD (amino acids 195-312) and of RIP (amino acids 261-372), fusedat their N-termini either with the FLAG octapeptide or the HA epitope(Field et al, 1988), were expressed, either alone or in mixtures of twoin HeLa cells and metabolically labeled with [³⁵S]-Cys and [³⁵S]-Met.Cross-immunoprecipitation of the co-expressed proteins was performedusing the indicated antibodies. The proteins were analyzed bySDS-polyacrylamide gel electrophoresis (15% acrylamide), followed byauto radiography. In cell lysates containing MORT-1 and RIPco-immunoprecipitation of both proteins could be obtained usingantibodies against either one of them. However, in lysates containingTRADD and REP, co-immunoprecipitation of the two proteins was observedonly when using antibody against RIP, and in lysates containing TRADDand MORT-1—only with an antibody against TRADD, apparently due to sterichindrance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to novel proteins orpeptides which are capable of binding to one or more ‘death domain’motifs of ‘death domain’ motif-containing proteins by virtue ofrecognizing sequence features common to the ‘death domain’ motifs withinthese proteins. Hence the ‘death domain’ motif binding proteins orpeptides are considered as mediators or modulators of this group of‘death domain’ motif containing proteins. This group of ‘death domain’motif-containing proteins includes: (i) members of the TNF/NGF receptorfamily such as, for example, p55 TNF-R, FAS-R (Fas/APO1) and the lowaffinity NGF receptor (NGF-R); (ii) other related proteins such as, forexample, the recently discovered protein called MORT-1 (or HF1) (for“Mediator of Receptor-Mediated Toxicity”) which, amongst itscharacteristics, is capable of self-association and specific binding tothe intracellular domain of FAS-R; as well as (iii) apparentlynon-related proteins such as, for example, the cytoskeletal proteinankyrin 1. The ‘death domain’ motif and some of its characteristics hasbeen disclosed in respect of the p55 TNF-R, FAS-R and MORT-1 in theco-pending Israel Application Nos. 109632, 111125, 112002 and 112692.The ‘death domain’ motif present in NGF-R and ankyrin 1 has beendiscovered in accordance with the present invention (see Example 1).

In the above noted co-pending applications there is described a numberof proteins capable of binding specifically to the intracellular domainsof p55-TNF-R and/or FAS-R, which proteins include MORT-1. However, incontrast, the present invention concerns, in this one aspect thereof,proteins or peptides which specifically bind to the ‘death domain’ motifof one or more of the above mentioned proteins belonging to the groupcharacterized by having such a ‘death domain’ motif, thebinding/interaction between the proteins or peptides of the inventionand the ‘death domain’ motif being by virtue of sequence features commonto the various ‘death domain’ motifs. Hence, the proteins or peptides ofthe invention are characterized by being capable of modulating ormediating the activity of one or more of the members of this group ofproteins by recognizing features common to the ‘death domain’ motifs.

Accordingly, included in the present invention is a large group ofproteins or peptides which bind to the various ‘death domain’ motifs, inwhich some of the proteins or peptides bind specific ‘death domain’motifs of specific proteins or receptors, while others bind more thanone such motif of more than one such protein/receptor. From FIG. 1 itarises that common sequence features of the ‘death domain’ motifs in‘death domain’ motif-containing proteins such as p55 TNF-R, FAS-F,NGF-R, MORT-1 and ankyrin 1 include common amino acid residues (residuesmarked within boxes such as the W (tryptophan), L (leucine), I(isoleucine), A (alanine), D (aspartic acid) and E (glutamic acid), aswell as T (threonine), R (arginine) and Y (tyrosine), at the locationshown in FIG. 1.

The proteins or peptides of the invention may be obtained as describedin the above noted co-pending patent applications (see also Example 3),by use of the yeast two-hybrid procedure in which the ‘death domain’motif of, for example, p55-TNF-F, FAS-R, MORT-1, NGF-R, ankyrin 1 willbe used as probes or ‘baits’ to isolate from genomic or cDNA libraries,clones expressing proteins or peptides capable of binding to one or moreof these ‘death domain’ motifs. Alternatively, a synthetic DNA sequencecan be synthesized in which there is included all of the common sequencefeatures of the ‘death domain’ motifs of p55-TNF-R, FAS-R, MORT-1,NGF-R, ankyrin 1 (see FIG. 1), to provide a common or “universal” ‘deathdomain’ motif sequence, which in turn can be used in the yeasttwo-hybrid procedure to isolate and identify clones from cDNA or genomiclibraries which encode proteins or peptides capable of binding to this‘death domain’ motif sequence.

Other approaches for obtaining the proteins and peptides of theinvention include the well known standard procedures such as, forexample, affinity chromatography in which, for example, peptides orprotein fragments having the ‘death domain’ motif sequence of p55 TNF-R,FAS-R, MORT-1, NGF-R and ankyrin 1, or a synthetically produced ‘deathdomain’ motif peptide having common sequence features of all theaforesaid ‘death domain’ motifs (see FIG. 1), are attached to thechromatography substrate or matrix and are brought into contact withcell extracts or lysates (of human/mammalian origin) and therebyproteins or peptides are isolated which are capable of binding to one ormore of these ‘death domain’ motifs. Likewise, other standard chemicaland recombinant DNA procedures usually employed for isolating proteinsor peptides capable of binding to a specific amino acid sequence (‘deathdomain’ motif sequence) can be employed to obtain the proteins andpeptides of the invention.

Thus, the present invention also concerns the DNA sequences encoding theproteins and peptides of the invention and the proteins and peptidesencoded by these sequences.

Moreover, the present invention also concerns the DNA sequences encodingbiologically active analogs and derivatives of these proteins andpeptides of the invention, and the analogs and derivatives encodedthereby. The preparation of such analogs and derivatives is by standardprocedure (see for example, Sambrook et al, 1989) in which in the DNAsequences encoding these proteins, one or more codons may be deleted,added or substituted by another, to yield analogs having at least a oneamino acid residue change with respect to the native protein. Acceptableanalogs are those which retain at least the capability of binding to the‘death domain’ motif of one or more of the members of the abovementioned group of ‘death domain’ motif-containing proteins, or whichcan mediate any other binding or enzymatic activity, e.g., analogs whichbind the ‘death domain’ motif but which do not signal, i.e., do not bindto a further downstream receptor, protein or other factor, or do notcatalyze a signal-dependent reaction. In such a way analogs can beproduced which have a so-called dominant-negative effect, namely, ananalog which is defective either in binding to the, ‘death domain’ motifor in subsequent signaling following such binding. Such analogs can beused, for example, to inhibit the TNF, FAS-ligand-, NGF-R-mediated,MORT-1-mediated and ankyrin 1-mediated effect by competing with thenatural IC-binding proteins.

Likewise, so-called dominant-positive analogs may be produced whichwould serve to enhance, for example, the TNF, FAS ligand.NGF-R-mediated, MORT-1-mediated and ankyrin 1-mediated effect. Thesewould have the same or better ‘death domain’ motif-binding propertiesand the same or better signaling properties of the natural ‘deathdomain’ motif-binding proteins. Similarly, derivatives may be preparedby standard modifications of the side groups of one or more amino acidresidues of the proteins, or by conjugation of the proteins to anothermolecule, e.g., an antibody, enzyme, receptor, etc., as are well knownin the art.

The new ‘death domain’ motif-binding proteins and peptides of theinvention, e.g., the proteins and peptides capable of binding one ormore of the ‘death domain’ motifs of p55 TNF-R, FAS-R, MORT-1, NGF-R andankyrin 1, as well as RIP and TRADD, have a number of possible uses, forexample:

(i) They may be used to mimic or enhance the function of TNF or FAS-Rligand, or the functions mediated by NGF-R, MORT-1, RIP, TRADD andankyrin 1 or other proteins containing the ‘death domain’ motif, insituations where such an enhanced effect is desired such as inanti-tumor, anti-inflammatory, or anti-HIV or other disease/disorderapplications where the enhanced activity is desired. In this case theproteins or peptides may be introduced to the cells by standardprocedures known per se. For example, as the proteins or peptides arerequired to act intracellularly, i.e., bind/interact withintracellularly located ‘death domain’ motifs and it is desired thatthey be introduced only into the cells where their effect is wanted, asystem for specific introduction of these proteins into the cells isnecessary. One way of doing this is by creating a recombinant animalvirus, e.g., one derived from Vaccinia, to the DNA of which thefollowing two genes will be introduced: the gene encoding a ligand thatbinds to cell surface proteins specifically expressed by the cells,e.g., ones such as the AIDs (HIV) virus gp120 protein which bindsspecifically to some cells (CD4 lymphocytes and related leukemias), or aligand that binds specifically to erythrocytes or nervous tissue (in thecase of ankyrin 1), or a ligand binding specifically to cellscharacterized by expressing other members of the ‘death domain’motif-containing group of proteins, e.g., those expressing MORT-1, RIP,TRADD, or any other ligand that binds specifically to cells carrying aTNF-R, FAS-R, or NGF-R such that the recombinant virus vector will becapable of binding such cells; and the gene encoding the new ‘deathdomain’ motif-binding protein or peptide. Thus, expression of thecell-surface-binding protein on the surface of the virus will target thevirus specifically to the tumor cell, HIV-infected cells or other cells,following which the ‘death domain’ motif-binding protein or peptideencoding sequence will be introduced into the cells via the virus, andonce expressed in the cells will result in enhancement of, for example,the TNF, FAS-R ligand, NGF-R-mediated, MORT-1-mediated, RIP- andTRADD-mediated, or ankyrin 1-mediated effect leading to, for example,the death of the tumor cells or other TNF-R- or FAS-R-carrying cells itis desired to kill. Construction of such recombinant animal virus is bystandard procedures (see for example, Sambrook et al, 1989). Anotherpossibility is to introduce the sequences of the new proteins orpeptides in the form of oligonucleotides which can be absorbed by thecells and expressed therein.

(ii) They may be used to inhibit, for example, the TNF, FAS-R ligand,NGF-R-mediated, MORT-1-mediated and ankyrin-1-mediated effect, e.g., incases such as tissue damage in septic shock, graft-vs-host rejection,acute hepatitis, or other diseases/disorders in which case it is desiredto block the TNF-induced TNF-R, FAS-R ligand induced FAS-R or NGFinduced NGF-R intracellular signaling or intracellular events mediatedby MORT-1, RIP, TRADD and ankyrin-1. In this situation it is possible,for example, to introduce into the cells, by standard procedures,oligonucleotides having the anti-sense coding sequence for these newproteins or peptides which would effectively block the translation ofmRNAs encoding these proteins and thereby block their expression andlead to the above noted desired inhibition of the effects mediated bythe ‘death domain’ motif-containing proteins.

Such oligonucleotides may be introduced into the cells using the aboverecombinant virus approach, the second sequence carried by the virusbeing the oligonucleotide sequence. Another possibility is to useantibodies specific for these proteins or peptides to inhibit theirintracellular signaling activity (via their binding to the ‘deathdomain’ motifs).

Yet another way of inhibiting the TNF FAS-R ligand, NGF-R-mediated,MORT-1-mediated, RIP- and TRADD-mediated, or ankyrin-1-mediated effector effects mediated by other ‘death domain’ motif-containing proteins,is by the recently developed ribozyme approach. Ribozymes are catalyticRNA molecules that specifically cleave RNAs. Ribozymes may be engineeredto cleave target RNAs of choice, e.g., the mRNAs encoding the newproteins or peptides of the invention. Such ribozymes would have asequence specific for the mRNA of choice and would be capable ofinteracting therewith (complementary binding) followed by cleavage ofthe mRNA, resulting in a decrease (or complete loss) in the expressionof the protein or peptide it is desired to inhibit, the level ofdecreased expression being dependent upon the level of ribozymeexpression in the target cell. To introduce ribozymes into the cells ofchoice any suitable vector may be used, e.g., plasmid, animal virus(retrovirus) vectors, that are usually used for this purpose (see also(i) above, where the virus has, as second sequence, a cDNA encoding theribozyme sequence of choice). Moreover, ribozymes can be constructedwhich have multiple targets (multi-target ribozymes) that can be used,for example, to inhibit the expression of one or more of the proteins orpeptides of the invention. (For reviews, methods, etc. concerningribozymes see Chen et al, 1992, Zhao et al, 1993; Shore et al, 1993,Joseph et al, 1993; Shimayama et al, 1993; Cantor et al, 1993; Barinaga,1993; Crisell et al, 1993 and Koizurni et al, 1993).

(iii) They may be used to isolate, identify and clone other proteins orpeptides which are capable of binding to them, e.g., other proteins orpeptides involved in the intracellular signaling process that aredownstream of the ‘death domain’ motif-containing proteins. In thissituation, these options, namely, the DNA sequences encoding them may beused in the yeast two-hybrid system (see Example 2, below) in which thesequence of these proteins or peptides will be used as “baits” toisolate, clone and identify from cDNA or genomic DNA libraries othersequences (“preys”) encoding proteins which can bind to these new ‘deathdomain’ motif-binding proteins. In the same way, it may also bedetermined whether the specific proteins or peptides of the presentinvention, namely, those which bind to the ‘death domain’ motif of p55TNF-R, FAS-R, NGF-R, MORT-1 and ankyrin can bind to yet other receptorsor proteins. Moreover, this approach may also be taken to determinewhether the proteins or peptides of the present invention are capable ofbinding to other known receptors or proteins in whose activity they mayhave a functional role, i.e., other as yet unidentified ‘death domain’motif-containing receptors or proteins.

(iv) The new proteins may also be used to isolate, identify and cloneother proteins of the same class, i.e., those binding to ‘death domain’motifs of the various receptors or proteins listed above or tofunctionally related receptors or proteins, and involved in theirmodulation/mediation. In this application the above noted yeasttwo-hybrid system may be used, or there may be used a recently developed(Wilks et al, 1989) system employing non-stringent southernhybridization followed by PCR cloning. In the Wilks et al publication,there is described the identification and cloning of two putativeprotein-tyrosine kinases by application of non-stringent southernhybridization followed by cloning by PCR based on the known sequence ofthe kinase motif, a conceived kinase sequence. This approach may beused, in accordance with the present invention using the sequences ofthe new proteins or peptides to identify and clone those of related‘death domain’ motif-binding proteins or peptides also capable ofbinding to ‘death domain’ motif-containing receptors or proteins.

(v) Yet another approach to utilizing the new proteins of the inventionis to use them in methods of affinity chromatography to isolate andidentify other proteins or factors to which they are capable of binding,e.g., other receptors related to TNF-Rs (TNF/NGF receptor superfamily)or other proteins or factors (e.g., related to MORT-1, ankyrin 1)involved in the intracellular signaling or structural regulationprocess. In this application, the proteins of the present invention, maybe individually attached to affinity chromatography matrices and thenbrought into contact with cell extracts or isolated proteins or factorssuspected of being involved in the intracellular signaling or structuralregulation process. Following the affinity chromatography procedure, theother proteins or factors which bind to the new proteins of theinvention, can be eluted, isolated and characterized.

(vi) As noted above, the new proteins or peptides of the invention mayalso be used as immunogens (antigens) to produce specific antibodiesthereto. These antibodies may also be used for the purposes ofpurification of the new proteins or peptides either from cell extractsor from transformed cell lines producing them. Further, these antibodiesmay be used for diagnostic purposes for identifying disorders related toabnormal functioning of, for example, the TNF, FAS-R ligand, NGF-R,MORT-1 or ankyrin 1 system, e.g., overactive or underactive TNF- orFAS-R ligand-induced cellular effect or NGF-R-, MORT-1- or ankyrin1-mediated cellular effects. Thus, should such disorders be related to amalfunctioning intracellular signaling or structural regulation systeminvolving the new proteins or antibodies, such antibodies would serve asan important diagnostic tool.

In another aspect, the present invention relates to complementarypeptides which may be synthesized by well known standard procedures ofthe art, that are capable of binding or interacting specifically withone or more of the ‘death domain’ motifs of the above mentioned group of‘death domain’ motif-containing proteins. These complementary peptideswill be synthesized using, for example, the ‘death domain’ motifsequences of p55-TNF-R, FAS-R, MORT-1, RIP, TRADD, NGF-R, ankyrin 1, assubstrates and synthesizing by standard chemical means peptides ofsequence that are complementary to these ‘death domain’ motif sequences.A suitable complementary peptide is one that will be capable of bindingto one or more of these ‘death domain’ motifs and thereby being capableof modulating or mediating the activity of ‘death domain’motif-containing proteins.

The complementary peptides may be generated using as substrate one ormore of the ‘death domain’ motif sequences set forth in FIG. 1 or may begenerated using a synthetic peptide (see above) which has a sequenceinclusive of all of the common sequence features of the known ‘deathdomain’ motif sequences, e.g., the above mentioned amino acid residuesW, L, I, A, D, E, T, R and Y.

The so-generated complementary peptides, and likewise, DNA sequencesencoding them, which may be readily produced by standard procedures, maybe employed, as noted above in any one of uses (i)-(vi), i.e., toenhance (gain-of-function) or inhibit the activity of proteins orreceptors containing a ‘death domain’ motif, or may be used to generatespecific antibodies thereto for modulation/mediation, isolation ordiagnostic purposes.

It should also be noted that included in the present invention are theantibodies (and their uses) specific to the proteins and peptides of theinvention including the complementary peptides, as well as antibodiesspecific to the ‘death domain’ motif peptides themselves, e.g., thosepeptides shown in FIG. 1 which are the ‘death domain’ motifs ofp55-TNF-R, FAS-R, MORT-1, NGF-R, ankyrin 1 and other proteins containingthe ‘death domain’ motif. These antibodies may be used for directlymodulating/mediating the activity of proteins or receptors containing‘death domain’ motifs or for isolation, identification andcharacterization (including diagnostic applications, as noted above) ofother proteins and receptors containing such ‘death domain’ motifs.

As regards the antibodies mentioned herein throughout, the term“antibody” is meant to include polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments thereof provided by any known technique, such as,but not limited to enzymatic cleavage, peptide synthesis or recombinanttechniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which populations containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler et al(1975); U.S. Pat. No. 4,376,110; Harlow et al, (1988) and Colligan etal, (1992, 1993), the contents of which references are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof. A hybridoma producing a mAb of the present invention may becultivated in vitro, in situ or in vivo. Production of high titers ofmabs in vivo or in situ makes this the presently preferred method ofproduction.

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al, (1984); Morrison et al, (1984); Boulianne et al, (1984);Cabilly et al, European Patent Application 125023 (1984); Neuberger etal (1985); Taniguchi et al, European Patent Application 171496 (1985);Morrison et al, European Patent Application 173494 (1986); Neuberger etal, PCT Application WO 8601533 (1986); Kudo et al, European PatentApplication 184187 (1986); Sahagan et al, (1986); Robinson et al, PCTApplication WO 87/02671 (1987); Liu et al, (1987); Sun et al, (1987);Better et al, (1988); and Harlow et al (1988). These references areentirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb with the mAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880, which is herein entirely incorporated byreference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the ‘death domain’ motif-containingpeptides, ‘death domain’-binding proteins or peptides, or ‘deathdomain’-binding complementary peptides, analogs or derivatives thereofof the invention may be used to induce anti-Id antibodies in suitableanimals, such as BALB/c mice. Spleen cells from such immunized mice areused to produce anti-Id hybridomas secreting anti-Id mAbs. Further, theanti-Id mAbs can be coupled to a carrier such as keyhole limpethemocyanin (KLH) and used to immunize additional BALB/c mice. Sera fromthese mice will contain anti-anti-Id antibodies that have the bindingproperties of the original mAb specific for an epitope of the aboveproteins, peptides, analogs or derivatives.

The anti-Id mAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as GRBprotein-α.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)2, whichare capable of binding antigen. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al, 1983).

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of the ‘death domain’-binding proteins or peptidesaccording to the methods disclosed herein for intact antibody molecules.Such fragments are typically produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting, with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the presentinvention may be used to quantitatively or qualitatively detect the‘death domain’ motif-binding proteins or peptides (includingcomplementary peptides) in a sample or to detect presence of cells whichexpress the ‘death domain’ motif-binding proteins or peptides of thepresent invention. This can be accomplished by immunofluorescencetechniques employing a fluorescently labeled antibody (see below)coupled with light microscopic, flow cytometric, or fluorometricdetection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of ‘death domain’motif-binding proteins or peptides of the present invention. In situdetection may be accomplished by removing a histological specimen from apatient, and providing the labeled antibody of the present invention tosuch a specimen. The antibody (or fragment) is preferably provided byapplying or by overlaying the labeled antibody (or fragment) to abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the ‘death domain’ motif-bindingproteins or peptides, but also its distribution on the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

Such assays for ‘death domain’ motif-binding proteins of the presentinvention typically comprises incubating a biological sample, such as abiological fluid, a tissue extract, freshly harvested cells such aslymphocytes or leukocytes, or cells which have been incubated in tissueculture, in the presence of a detectably labeled antibody capable ofidentifying the ‘death domain’ motif-binding proteins or peptides, anddetecting the antibody by any of a number of techniques well known inthe art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a detectably labeled antibody in accordance with thepresent invention, as noted above. The solid phase support or carriermay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on said solid support or carrier maythen be detected by conventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know many other suitable carriers for binding antibodyor antigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand use in an enzyme immunoassay (EIA). This enzyme, in turn, when laterexposed to an appropriate substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomeras, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactivity labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al (1978) with particular reference to the chapter entitled “AnIntroduction to Radioimmune Assay and Related Techniques” by Chard,incorporated by reference herein. The radioactive isotope can bedetected by such means as the use of a y counter or a scintillationcounter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²E, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthe contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

The new proteins and peptides of the invention once isolated, identifiedand characterized by any of the standard screening procedures, forexample, the yeast two-hybrid method, affinity chromatography, and anyother well known method known in the art, may then be produced by anystandard recombinant DNA procedure (see for example, Sambrook, et al,1989) in which suitable eukaryotic or prokaryotic host cells aretransformed by appropriate eukaryotic or prokaryotic vectors containingthe sequences encoding for the proteins. Accordingly, the presentinvention also concerns such expression vectors and transformed hostsfor the production of the proteins of the invention. As mentioned above,these proteins also include their biologically active analogs andderivatives, and thus the vectors encoding them also include vectorsencoding analogs of these proteins, and the transformed hosts includethose producing such analogs. The derivatives of these proteins are thederivatives produced by standard modification of the proteins or theiranalogs, produced by the transformed hosts.

In another aspect, the present invention relates to the use of thevarious different ‘death domain’ motifs or the synthetically produced“universal” ‘death domain’ motif (having structural features common tomany different ‘death domain’ motifs) as agents for enhancing (gain offunction) the intracellular effect mediated by the natural ‘deathdomain’ motif-containing proteins. In this aspect the ‘death domain’motifs will be used in the form of peptides containing all of the ‘deathdomain’ motif or active parts thereof and introduced into the cells asmentioned above (e.g., the vaccinia virus approach). In this regard itshould be noted that the term ‘death domain’ was coined following thediscovery (see the copending patent applications noted above) that thisregion of the intracellular domains of the p55 TNF-R and FAS-R was theregion involved in the ligand-independent self-association andcell-cytotoxicity induction mediated by these receptors. In fact, thefree ‘death domain’ of p55 TNF-R (p55DD) is capable of self-associatingand inducing cell cytotoxicity. Further, upon discovery of the MORT-1protein which is a FAS-R binding protein, it was also found that thisprotein is capable of self-association and inducing, in aligand-independent and FAS-R-independent manner, cytotoxic effects oncells. The MORT-1 protein was subsequently observed to contain a ‘deathdomain’ motif homologous to the ‘death domains’ or ‘death domain’ motifsof p55 TNF-R and FAS-R (see FIG. 1), which ‘death domain’ motif isinvolved in MORT-1 association with FAS-R and is associated with theMORT-1 protein's ability to induce cell cytotoxic effects.

Thus, using the ‘death domain’ motifs of proteins such as p55-TNF-R,FAS-R and MORT-1 and any other proteins involved in the induction ofcytotoxic effects, in the way described above, it is possible to enhancethe cell cytotoxic effects normally mediated by the naturally-occurringcounterparts of these proteins, i.e., it would be possible to enhancethe killing of cells such as tumor cells, HIV-infected and otherdiseased cells, the killing of which is usually mediated by p55 TNF-R,FAS-R, MORT-1, RIP or TRADD, by introducing into such cells the ‘deathdomain’ motifs of these receptors/proteins.

Moreover, it is also possible to produce analogs of these ‘death domain’motifs which will provide an even better enhancement of their action,i.e., enhanced cell cytotoxicity, these analogs having one or more aminoacids added, deleted or replaced with respect to the naturally occurringsequences.

In a similar fashion it is also possible by the means described hereinabove to introduce ‘death domain’ motifs or analogs thereof, of theNGF-R or ankyrin 1 into cells in which it is desired to enhance theintracellular effects mediated by NGF-R or ankyrin 1.

Likewise, the present invention also relates to the specific blocking ofthe effects mediated by the ‘death domain’ motif-containing proteins byblocking the activity of the ‘death domain’ motifs of these proteins,e.g., by the introduction of anti-sense oligonucleotides into cells (asmentioned above) which would block the expression of the ‘death domain’motifs.

In yet another aspect of the invention there is provided organiccompounds, e.g., heterocyclic compounds, which are capable ofspecifically binding to the ‘death domain’ motifs of one or more ‘deathdomain’ motif-containing proteins. These organic compounds are wellknown in the field of pharmaceuticals and are widely used as therapeuticagents which are capable of entering cells (hydrophobic/lipophiliccompounds) and binding various intracellular proteins or intracellularportions of transmembrane proteins and thereby exerting their effect.These organic compounds may be readily screened and identified by usingthe ‘death domain’ motifs of the ‘death domain’ motif-containingproteins, e.g., those of p55 TNF-R, FAS-R. NGF-R, MORT-1, ankyrin 1, instandard affinity chromatography procedures or other methods well knownin the art.

It should also be mentioned, that the ‘death domain’ motif consists ofboth general structural features common to all of the various suchmotifs, i.e., a common scaffold, as well as specific structuralfeatures, specific to each of the ‘death domain’ motifs. Accordingly, apreferred drug or pharmaceutically active molecule according to theinvention will contain, as active ingredient naturally occurringproteins or peptides; synthetically produced proteins or peptidesincluding complementary peptides; antibodies; or chemical compoundsobtained by screening or design, all of which are characterized by beingcapable of recognizing the general ‘death domain’ features and one ormore of the specific ‘death domain’ features.

The present invention also relates to pharmaceutical compositionscomprising recombinant animal virus vectors encoding the ‘death domain’motif-binding proteins or peptides or the ‘death domain’ motif sequencesthemselves, which vector also encodes a virus surface protein capable ofbinding specific target cell (e.g., cancer cells) surface proteins todirect the insertion of the ‘death domain’ motif-binding protein orpeptide sequences or the ‘death domain’ motif sequences into the cells.Likewise, the present invention also relates to pharmaceuticalcompositions comprising organic compounds capable of binding to ‘deathdomain’ motifs of ‘death domain’ motif-containing proteins.

The invention will now be described in more detail in the followingnon-limiting examples and the accompanying drawings

EXAMPLE 1 The ‘Death Domain’ Motif Common to the Receptors P55 TNF-RFAS-R and NGF-R and to the Proteins MORT-1 and Ankyrin 1

Upon the discovery of MORT-1 (see co-pending IL 109632, IL 112002 and IL112692) it was also discovered that MORT-1 contains a region havinghomology to the previously identified ‘death domains’ of p55 TNF-R andFAS-R (p55DD and FAS-DD, respectively), see IL 109632 and IL 111125.This surprising discovery of a ‘death domain’ motif in a previouslyunknown protein led to a search for the existence of such a ‘deathdomain’ motif in other proteins. Surprisingly, such a ‘death domain’motif was discovered in the low affinity NGF-R and in an apparentlyunrelated, cytoskeletal protein, ankyrin 1. The ‘death domain’ motifs ofall these different proteins share a remarkable homology as is set forthschematically in FIG. 1, which shows a sequence comparison of the ‘deathdomain’ motifs of the p55 TNF-R, FAS-R, MORT-1, low affinity NGF-R andthe C terminal part of the regulatory domain in ankyrin 1 (all of humanorigin). The homology of these ‘death domain’ motifs was defined by theLINEUP and PRETTY programs of the GCG package. Identical and similarresidues in three or more of the proteins are boxed. Gaps introduced tomaximize alignment are denoted by dots. The significance of thishomology was confirmed as follows: (a) multiple alignment of the ‘deathdomain’ motif sequences, using the HSSP program of the PredictProteinService (Sander et al, 1991) showed sequence identities of 21-38% andsequence similarities of 30-48%; (b) searching the Swiss-Prot data bankwith a profile created (using the PILEUP, LINEUP and PROFILEMAKEprograms of the GCG package) from consensuses of the ‘death domain’motif sequences in the known p55 R and FAS-R (human, mouse, rat), NGFreceptor (human, rat and chicken) and ankyrins (human and mouse ankyrin1 and the human ankyrins c and g) sequences and in MORT-1 yielded highscores only those sequences that were used for creating the profile(Zscores>8.5 for all of them in search with the “Bioaccelerator”Compugen, Israel).

The above homology search using the PredictProtein Service (PHDsec) andthe PRODOM program of the GCG package revealed significant similaritybetween a region of approximately 65 residues in MORT-1, within thatpart of the molecule that binds to FAS-R, and a region of that samelength within the ‘death domains’ of FAS-R and p55-R, (FIG. 1). Thispart of the ‘death domain’ also shows similarity to a region in theintracellular domain of the low-affinity NGF receptor (Johnson et al,1986), a receptor whose extracellular domain is known to contain anotherconserved sequence motif common to FAS-R, the TNF-Rs and other membersof the TNF/NGF receptor family. It also revealed a previously-unnoticedsimilarity between this part of the ‘death domain’ and a conservedregion in the ankyrins, which are structural proteins that linkspectrin-based membrane skeletal proteins to the cytoplasmic domains ofintegral plasma membrane proteins (Lux et al, 1990; Lambert et al,1993). That region is located in the N terminal part of the ankyrinregulatory domain, just upstream to that part of the domain whoseexpression is subject to modulation by alternative splicing, and belowthe spectrin-binding and membrane binding domains. (The latter domaincontains another known sequence motif the ‘ankyrin repeat’). The ‘deathdomain’ motif is distinct from the ankyrin repeat motif that is found inthe membrane binding domain of the ankyrins.

The finding of a ‘death domain’ motif in proteins having differentintracellular effects suggests that this motif plays a more general rolethan that implied in the name ‘death domain’, i.e., this motif occurs inreceptors such as p55 TNF-R, FAS-R and the related protein MORT-1 whichmediate cell cytotoxicity, as well as in the NGF-R which, when inducingdeath does so only in the absence of ligand (Rabizadeh et al, 1993) andin proteins such as the cytoskeletal ankyrins, not associated with cellcytotoxic effects. One kind of general activity of this ‘death domain’motif, found so far in three of the proteins containing it, i.e., FAS-R,p55 TNF-R and MORT-1 is the ability to self-associate or interact withother proteins that contain this motif.

The discovery of the ‘death domain’ motif in such a wide range ofdifferent proteins provides the way for obtaining (as noted herein aboveand in Example 2 below) proteins or peptides capable of binding to thedifferent (one or more) ‘death domain’ motifs, which proteins andpeptides may be used as modulators/mediators of a wide group ofregulatory proteins, be they cytokine receptors involved in cellcytotoxic (p55 TNF-R, FAS-R) or growth (NGF-R) effects or relatedproteins involved in cell cytotoxic effects (MORT-1) or regulatoryportions of structural proteins involved in the shape/conformationalregulation of cells (ankyrins). In a similar fashion, the ‘death domain’motifs of these various proteins may also be used directly formodulation/mediation of proteins containing such motifs.

EXAMPLE 2 Interaction of ‘Death Domains’ of Human p55—TNF-R, FAS-R,TRADD. MORT-1 and RIP Experimental Procedures

Two-Hybrid β-Galactosidase Expression Tests. cDNA inserts were cloned byPCR, either from the full-length cDNAs cloned previously in ourlaboratory, or from purchased cDNA libraries. Residue numbering in theproteins encoded by the cDNA inserts are as in the Swiss-Prot Data Bank.Point mutants were produced by oligonucleotide-directed mutagenesis(Kunkel, 1994). β-galactosidase expression in yeasts (SFY526 reporterstrain (Bartel et al, 1993)) transformed with these cDNAs in the pGBT-9and pGAD-GH vectors (DNA binding domain (DBD) and activation domain (AD)constructs, respectively) was assessed by a filter assay (Boldin et al,1995). When expressed in the pGAD-GH vector, RIP and its DD had somecytotoxic effect on the yeasts, manifested in a low yield of yeastcolonies. They did not have any such cytotoxic effect when expressed (toa lower extent) in the pGBT-9 vector.

Induced Expression, Metabolic Labeling and Immunoprecipitation ofProteins. Since the size similarity of the DDs makes it difficult todistinguish between them in gel electrophoresis, we chose to examine theinteraction of MORT-1, TRADD and RIP by co-expressing the full-lengthMORT-1 protein with the DDs of TRADD and RIP. The proteins, N-linked tothe FLAG octapeptide (Eastman-Kodak, New Haven, Conn.), or to aninfluenza hemagglutinin epitope (HA epitope, YPYDVPDYA (Field et al,1988)) were expressed in HeLa cells, using a tetracycline-controlledexpression vector, and labeled metabolically with [³⁵S]-Met (55 μCi/ml)and [³⁵S]-Cys (10 μCi/ml) (EXPRE³⁵S³⁵S Protein Labeling Mix, DuPont,Wilmington. DE), as described before (Boldin et al, 1995). The cellswere then lysed in RIPA buffer (1 ml/5×10⁵ cells) and the lysates wereprecleared by incubation with irrelevant rabbit antiserum (3 μl/ml) andProtein G Sepharose beads (Pharmacia, Uppsala, Sweden, 6-μl/ml).Immunoprecipitation was performed by 1 hour incubation at 4° C. of 0.3ml aliquots of lysate with mouse monoclonal antibodies (5 μg/aliquot)against the FLAG octapeptide (M2; Eastman Kodak). HA epitope (12CA5(Field et al, 1988)), or the p75 TNF-R (#9; (Bigda et al, 1994)) as acontrol, followed by an additional 1 hour incubation with Protein GSepharose beads (30 μl/aliquot). The immunoprecipitates were washed 3times with RIPA buffer and analyzed by SDS-polyacrylamide gelelectrophoresis.

Evaluation

The interactions of the DDs of human p55 TNF-R, FAS-R, TRADD, MORT-1 andRIP were evaluated first by a yeast two-hybrid test. The cDNAs encodingthese domains were expressed as fusion proteins with the Gal4 DNAbinding and activation domains (DBD and AD constructs) in the yeastSFY526 reporter strain, and the binding of these fusion proteins to eachother was assessed by determining β-galactosidase expression by theyeasts. The results of these tests are summarized in FIG. 1 andillustrated diagrammatically in FIG. 3.

The DDs of P55 TNF-R, FAS-R, TRADD and RIP were able to self-associate.The DD of MORT-1 lacked this ability, even though the full-length MORT-1protein does self-associate (Boldin et al, 1995), apparently through aninteraction that involves the region upstream of its DD.

The DD of TRADD bound to the DD of p55 TNF-R, but not to the bD ofFAS-R, while the DD of MORT-1 behaved in the converse fashion.

The DD of RIP, like the full-length RIP protein (Stanger et al, 1995),was able to bind both to the DDs of FAS-R and p55 TNF-R. Binding wassignificantly weaker, though, than that of the DDs of TRADD and MORT-1to these receptors. Although RIP was initially identified by virtue ofits binding in a two-hybrid screen to FAS-R (Stanger et al, 1995), thisbinding is quite weak, and could be observed only when the RIP DD washighly expressed in the yeasts, by introducing it into the AD construct.There was no measurable binding when the DD of RIP was introduced intothe DBD construct, which has a lower expression effectivity. A longerRIP insert, corresponding to amino acids 161-372 in the protein, did notbind more effectively to FAS-R (not shown).

Apart from their observed binding to the DDs of P55 TNF-R or FAS-R, theDDs of each of the three intracellular proteins tested bound also toeach other. These interactions were all effective. Notably, theeffectivity of binding of the DD of REP to the DDs of MORT-1 and TRADDwas significantly greater than that of its binding to the DDs of p55TNF-R and FAS-R.

A similar pattern of interaction was observed in the HF7c yeast reporterstrain, regularly used in inventors' laboratory for two-hybrid screens.Indeed, in a recent attempt to clone proteins that bind to MORT-1 by atwo-hybrid screen, it was found that a significant proportion of thecloned cDNAs encode TRADD or RIP (not shown).

In specificity tests for the two-hybrid assay, we did not observebinding of the DD motifs to any of a number of irrelevant proteins,including SNF 1, the intracellular domain of the human p75 TNF receptor,lamin, cycline D and the DD of the rat low-affinity NGF receptor (notshown). To further assess the binding specificity, we introduced pointmutations to the p55 TNF-R, FAS-R, MORT-1 and RIP DDs, at sitescorresponding to that of 1-225 in the mouse FAS-R sequence. Anaturally-occurring replacement mutation of this residue, found inlpr^(cg) mice, abolishes signaling by FAS-R (Itoh and NaQata, 1993;Watanabe-Fukunaga et al, 1992) as well as its interaction with MORT-1(Boldin et al, 1995; Chinnaiyan et al, 1995). Mutation of thecorresponding residues in the DDs of human p55 TNF-R (L351N) and FAS-R(V238N) had a similar effect. The mutated proteins were not able toself-associate, nor to bind to TRADD, MORT-1 or RIP. Also, introductionof a replacement mutation to the DD of RIP at the site corresponding tothat of the lpr^(cg) mutation (F308N) resulted in loss of its ability tobind to FAS-R, MORT-1 and TRADD, as well as to self-associate, althoughthe mutated protein bound to the normal RIP DD. On the other hand, inMORT-1 the lpr^(cg) like mutation (V121N) had only a limited effect. Itresulted in less effective binding to FAS-R which, for some reason, wasobserved only when the mutated protein was introduced into the ADconstruct but not in the DBD construct.

To test whether the interactions observed between TRADD, MORT-1 and RIPin the yeast two-hybrid tests occur also in eukaryotic cells, weco-expressed MORT-1 and the DDs of TRADD and RIP within transfected HeLacells and attempted to immunoprecipitate them from the cell lysates.Immunoprecipitation resulted in precipitation of the co-expressedproteins, indicating that they bind to each other within the HeLa Cells(FIG. 4).

Although the evidence is still largely indirect, TRADD, MORT-1 and RIPappear to play important roles in the initiation of the cytocidal effectof p55 TNF-R and FAS-R (Cleveland et al, 1995). The binding of theseproteins to the receptors, which occurs through their DDs, apparently isrequired for their contribution to the signaling. A recent study showingthat stimulation of FAS-R in cells evokes binding of MORT-1 to thisreceptor suggests that the DD interactions observed within transfectedyeasts also occur within the mammalian cells, and take part in theprocess of signaling induction (Kischkel et al, 1995). Although the DDsof all the proteins examined have the ability to bind to other DDs,there is clear specificity in this interaction. The DD of TRADD binds tothat of p55 TNF-R, but not to the DD of FAS-R. The DD of MORT-1 binds tothe DD of FAS-R, but does not bind to the DD of p55 TNF-R. Thisspecificity in the action of proteins that take part in the signalingactivity of p55 TNF-R and FAS-R may well contribute to the differencesin function of the two receptors.

In addition to their differential binding to the DDs of p55 TNF-R andFAS-R, the DDs of TRADD and MORT-1 also are able to bind effectively toeach other, and both are capable of binding to the DD of REP moreeffectively than do the DDs of FAS-R or p55 TNF-R. Thus, even thoughdistinct, the signaling cascades affected by TRADD and MORT-1 may wellturn to be coordinated through their mutual interactions. The nature ofthis coordination may vary, depending on the way in which the differentinteractions of the DD in a given protein affect each other. Theseinteractions may occur together or be exclusive; they may also modulateeach other. One possible way for such modulation is indicated by theoccurrence in RIP of sequence motifs characteristic of protein kinases.If this protein indeed possesses protein kinase activity, it may turn tobe capable of phosphorylating MORT-1 and TRADD upon binding to them,thereby modulating their function. One plausible consequence of theassociation of TRADD and MORT-1, and of the binding of RIP to bothproteins, is integration of their effects, at least in part. Thisintegration may account for the fact that cell death induction by p55TNF-R and FAS-R exhibit, alongside distinct features, also certainsimilarities; this could result also in sharing of other activities ofthe two receptors.

EXAMPLE 3 Cloning and Isolation of Proteins which Bind to the ‘DeathDomain’ Motifs of ‘Death Domain’ Motif-Containing Proteins

To isolate proteins interacting with the ‘death domain’ motifs of ‘deathdomain’ motif-containing proteins, for example, the ‘death domain’motifs of p55 TNF-R, FAS-R, NGF-R, MORT-1 and ankyrin 1, the yeasttwo-hybrid system (Fields et al, 1989) may be used as described inco-pending Israel patent application Nos. 109632, 112002 and 112692.Briefly, this two-hybrid system is a yeast-based genetic assay to detectspecific protein-protein interactions in vivo by restoration of aeukaryotic transcriptional activator such as Gal4 that has two separatedomains, a DNA binding and an activation domain, which domains whenexpressed and bound together to form a restored Gal4 protein, is capableof binding to an upstream activating sequence which in turn activates apromoter that controls the expression of a reporter gene, such as lacZor HIS3, the expression of which is readily observed in the culturedcells. In this system the genes for the candidate interacting proteinsare cloned into separate expression vectors. In one expression vectorthe sequence of the one candidate protein is cloned in phase with thesequence of the Gal4 DNA-binding domain to generate a hybrid proteinwith the Gal4 DNA-binding domain, and in the other vector the sequenceof the second candidate protein is cloned in phase with the sequence ofthe Gal4 activation domain to generate a hybrid protein with theGal4-activation domain. The two hybrid vectors are then co-transformedinto a yeast host strain having a lacZ or HIS3 reporter gene under thecontrol of upstream Gal4 binding sites. Only those transformed hostcells (co-transformants) in which the two hybrid proteins are expressedand are capable of interacting with each other, will be capable ofexpression of the reporter gene. In the case of the lacZ reporter gene,host cells expressing this gene will become blue in color when X-gal isadded to the cultures. Hence, blue colonies are indicative of the factthat the two cloned candidate proteins are capable of interacting witheach other.

Using this two-hybrid system, the ‘death domain’ motifs may be cloned,separately, into the vector pGBT9 (carrying the Gal4 DNA-bindingsequence, provided by CLONTECH, USA, see below), to create fusionproteins with the Gal4 DNA-binding domain. Once the sequence of the‘death domain’ motif is known, e.g., those shown in FIG. 1, the DNAsequence encoding these motifs may be readily isolated and cloned, bystandard procedures into the pGBT9 vector utilizing the vector'smultiple cloning site region (MCS).

The above hybrid (chimeric) pGBT9 vectors can then be co-transfected(separately, one co-transfection with each ‘death domain’motif-containing hybrid together with a cDNA or genomic DNA library fromhuman or other mammalian origin, e.g., a cDNA library from human HeLacells cloned into the pGAD GH vector, bearing the Gal4 activatingdomain, into the HF7c yeast host strain (all the above-noted vectors,pGBT9 and pGAD GH carrying the HeLa cell cDNA library, and the yeaststrain are purchasable from Clontech Laboratories, Inc., USA, as a partof MATCHMAKER Two-Hybrid System, #PT1265-1). The co-transfected yeastsare then selected for their ability to grow in medium lacking Histidine(HIS⁻ medium), growing colonies being indicative of positivetransformants. The selected yeast clones were then tested for theirability to express the lacZ gene, i.e., for their LAC Z activity, andthis by adding X-gal to the culture medium, which is catabolized to forma blue colored product by β-galactosidase, the enzyme encoded by thelacZ gene. Thus, blue colonies are indicative of an active lacZ gene.For activity of the lacZ gene, it is necessary that the Gal4transcription activator be present in an active form in the transformedclones, namely that the Gal4 DNA-binding domain encoded by one of theabove hybrid vectors be combined properly with the Gal4 activationdomain encoded by the other hybrid vector. Such a combination is onlypossible if the two proteins fused to each of the Gal4 domains arecapable of stably interacting (binding) to each other. Thus, the His⁺and blue (LAC Z⁺) colonies that are isolated are colonies which havebeen co-transfected with a vector encoding a ‘death domain’ motif and a‘vector encoding a protein product of, for example, human HeLa cellorigin that is capable of binding stably to a ‘death domain’ motif.

The plasmid DNA from the above His⁺, LAC Z⁺ yeast colonies can then beisolated and electroporated into E. coli strain HB101 by standardprocedures followed by selection of Leu+ and Ampicillin resistanttransformants, these transformants being the ones carrying the hybridpGAD GH vector which has both the Amp® and Leu² coding sequences. Suchtransformants therefore are clones carrying the sequences encoding newlyidentified proteins or peptides capable of binding to the ‘death domain’motifs. Plasmid DNA was then isolated from these transformed E. coli andretested by:

(a) retransforming them with the original ‘death domain’motif-containing hybrid plasmids into yeast strain HF7 as set forthhereinabove. As controls, vectors carrying irrelevant protein encodingsequences, e.g., pACT-lamin or pGBT9 alone can be used forco-transformation with the ‘death domain’ motif-binding protein orpeptide encoding plasmids. The co-transformed yeasts can then be testedfor growth on His⁻ medium alone, or with different levels of3-an-˜notria-zole; and

(b) retransforming the plasmid DNA and original ‘death domain’ motifhybrid plasmids and control plasmids described in (a) into yeast hostcells of strain SFY526 and determining the LAC Z+activity (effectivelyof β-gal formation, i.e., blue color formation). It should be noted thatthe above noted β-galactosidase (β-gal) expression tests can also bedone by a standard filter assay.

EXAMPLE 4 Assessment of the Involvement of Sequence FeaturesCharacteristic of the ‘Death Domain’ Motif in the Binding of the ClonedProteins

The cDNA encoding, the protein that contains the ‘death domain’ motifwill be mutated at the various amino acids that constitute this motif.For example, tryptophan 380 in the intracellular domain of the humanlow-affinity nerve growth factor receptor (NGF-R) will be replaced withalanine. Such mutation can be performed, for example, by the Kunkeloligonucleotide-directed mutagenesis procedure. The mutated, as well asthe wild-type proteins, can be produced in bacteria as fusions withGlutathione S-transferase (GST). The binding of the cloned protein invitro to the GST fusion with the mutated NGF-R will be compared to itsbinding to the GST-wild type NGF-R intracellular domain fusion.Abolition of the binding by the mutation will indicate that the clonedprotein indeed recognizes sequence features that are involved in the‘death domain’ motif. A similar approach will be taken to assess theinvolvement of the sequence features characteristic of the ‘deathdomain’ in the function of other reagents that interact with proteinscontaining this motif, namely antibodies, peptides or organic compounds(See Example 4).

EXAMPLE 5 Design of Drugs That Affect ‘Death Domain’ Motif-ContainingProteins by Virtue of Their Ability to Interact with the ‘Death Domain’

Organic molecules or peptides that interact with the ‘death domain’motif of one of the proteins containing this motif will be definedeither by screening or by design. Further changes will then beintroduced to this molecule to increase the effectivity of itsinteraction with that specific ‘death domain’ and the ability of thedesigned compound to affect (enhance or interfere with) the function ofthe protein containing the ‘death domain’. Once creating such a moleculeand defining the sequence feature of the ‘death domain’ which itrecognizes (see Example 3) as well as the conformational features of the‘death domain’ involved in this recognition (by NMR, X-raycrystallography, etc.), this knowledge can be applied as a startingpoint for designing drugs that will affect other proteins containing the‘death domain’ motif. To do so, one should introduce to the designedpeptide or organic molecule, besides structural features that allowrecognition of those structural features that are common to the ‘deathdomain’, also structural features that will dictate specific recognitionof the specific ‘death domain’ containing protein.

EXAMPLE 6 Analysis of the Biological Activity of the ‘Death Domain’Motif Binding Proteins, Peptides, Antibodies or Organic Molecules

Once the ‘death domain’ motif binding proteins or peptides have beenisolated, e.g., by the procedure of Example 1, they can be tested fortheir biological activity. In copending applications IL 109632, IL111125, IL 112002 and IL 112692, there is described one such procedurewhich assays the effect of intracellular domain binding proteins of thecytotoxic effects mediated by the p55 TNF-R, FAS-R and MORT-1 (HF1).

Thus, using similar procedures it is possible to determine, firstly, theability of such ‘death domain’ motif-binding proteins or peptides toassociate in vitro with ‘death domain’ motif-containing proteins such asp55 TNF-R, FAS-R, NGF-R, MORT-1, REP, TRADD and ankyrin 1; and secondlyto assess in vivo, using standard cell cytotoxicity assays, whether such‘death domain’ motif binding proteins or peptides are capable ofenhancing or inhibiting the cell cytotoxicity induced by such receptorsas p55 TNF-R or FAS-R or proteins such as MORT-1.

Likewise, the same tests may also be applied to assay organic compounds(obtained by screening or design, see Example 4); synthetically producedpeptides (see Example 4); and antibodies, capable of binding to ‘deathdomain’ motifs.

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1. A modulator of regulatory cellular events occurring intracellularlythat are mediated by regulatory proteins containing a ‘death domain’motif which is a regulatory portion of said proteins, said modulatorbeing capable of interacting with one or more of the ‘death domain’motifs contained in said regulatory proteins and affecting theregulatory action of one or more of said regulatory proteins.
 2. Amodulator according to claim 1, wherein said modulator is selected fromthe group comprising naturally-derived ‘death domain’ motif-bindingproteins and peptides and analogs and derivatives thereof capable ofinteracting with one or more of said ‘death domain’ motifs.
 3. Amodulator according to claim 1, wherein said modulator is selected fromthe group of synthetically produced complementary peptides, synthesizedby using as substrates the ‘death domain’ motif sequences of saidregulatory proteins containing ‘death domain’ motifs, said complementarypeptides being capable of interacting with one or more of said ‘deathdomain’ motifs.
 4. A modulator according to claim 1, wherein saidmodulator is selected from the group consisting of organic compoundscapable of interacting with one or more of said ‘death domain’ motifs,said organic compounds being derived from known compounds and selectedby using said ‘death domain’ motifs as a substrate in a binding assay,or being synthesized using said ‘death domain’ motifs as a substrate fordesigning and synthesizing said organic compounds.
 5. A modulatoraccording to claim 1, wherein said modulator is selected from the groupconsisting of peptides or polypeptides derived from naturally-occurring‘death domain’ motif sequences, said peptides or polypeptides beingcapable of interacting with one or more of said ‘death domain’ motifs,and analogs and derivatives of said peptides or polypeptides capable ofinteracting with one or more of said ‘death domain’ motifs.
 6. Amodulator according to claim 1, wherein said modulator is furthercharacterized by being capable of recognizing the general ‘death domain’motif sequence features common to the ‘death domain’ motifs of ‘deathdomain’ motif containing proteins, and being capable of recognizing oneor more of the specific ‘death domain’ motifs of said proteins, saidspecific sequence features being specific to each ‘death domain’ motifsequence of each of said proteins.
 7. A modulator according to claim 1,wherein said modulator is capable of interacting with one or more of the‘death domain’ motifs contained within the proteins belonging to thegroup comprising p55 TNF-R, FAS-R, NGF-R, MORT-1, RIP, TRADD andankyrin
 1. 8. A modulator according to claim 7, wherein said modulatoris further characterized by being capable of interacting with sequencefeatures which the ‘death domain’ motifs of said group of proteins havein common, said common sequence features comprising the group of commonamino acid residues W (tryptophan), L (leucine), I (isoleucine), A(alanine), D (aspartic acid), E (glutamic acid), T (threonine), R(arginine) and Y (tyrosine) at the location within said ‘death domain’motifs of SEQ ID NOs:1-5.
 9. A DNA sequence encoding a modulator being aprotein, peptide or polypeptide or an analog of any thereof, accordingto claim
 1. 10. A DNA sequence according to claim 9, encoding anaturally-derived protein or peptide, selected from the group consistingof: (a) a cDNA sequence derived from the coding region of a native‘death domain’ motif-binding protein or peptide; (b) DNA sequencescapable of hybridization to a sequence of (a) under moderately stringentconditions and which encode a biologically active ‘death domain’motif-binding protein or peptide; and (c) DNA sequences which aredegenerate as a result of the genetic code to the DNA sequences definedin (a) and (b) and which encode a biologically active ‘death domain’motif-binding protein or peptide.
 11. A sequence according to claim 9,encoding a ‘death domain’ motif-binding protein or peptide capable ofbinding to the ‘death domain’ motif of one or more of the proteinsselected from the group consisting of p55 TNF-R, FAS-R, NGF-R, MORT-1,RIP, TRADD and ankyrin
 1. 12. A DNA sequence according to claim 9,encoding a peptide or polypeptide derived from the naturally-occurring‘death domain’ motif sequence of the ‘death domain’ motif-containingproteins.
 13. A DNA sequence according to claim 12, encoding a peptideor polypeptide derived from the ‘death domain’ motif sequence of any oneof the proteins of the group comprising p55 TNF-R, FAS-R NGF-R, MORT-1,RIP, TRADD and ankyrin
 1. 14. A protein, peptide or polypeptide, or ananalog of any one thereof, encoded by a DNA sequence according to claim9, said protein, peptide, polypeptide or analog being capable of bindingto or interacting with one or more of the ‘death domain’ motifs of oneor more ‘death domain’ motif-containing proteins.
 15. A vectorcomprising a DNA sequence according to claim
 9. 16. A vector accordingto claim 15, which is capable of being expressed in a eukaryotic hostcell.
 17. A vector according to claim 15, which is capable of beingexpressed in a prokaryotic host cell.
 18. Transformed eukaryotic orprokaryotic host cells containing a vector according to claim
 15. 19. Amethod for producing a protein, peptide, polypeptide or analog which iscapable of binding to or interacting with one or more of the ‘deathdomain’ motifs of one or more ‘death domain’ motif-containing proteins,comprising growing the transformed host cells according to claim 18under conditions suitable for the expression of said protein, peptide,polypeptide or analog, effecting post-translational modifications ofsaid protein, peptide, polypeptide or analog as necessary for obtentionthereof, and extracting said expressed protein, peptide, polypeptide oranalog from culture medium of said transformed cells or from cellextracts of said transformed cells.
 20. A method for the modulation ofthe TNF or FAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R,or the functions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin1 or by other proteins containing a ‘death domain’ motif, comprisingtreating said cells with one or more proteins, peptides, polypeptides oranalogs selected from the group consisting of the proteins, peptides,polypeptides or analogs according to claim 14, all being capable ofbinding to or interacting with the ‘death domain’ motif and modulatingthe activity of said ‘death domain’ motif-containing proteins, whereinsaid treating of said cells comprises introducing into said cells saidone or more proteins, peptides, polypeptides or analogs in a formsuitable for intracellular introduction thereof, or introducing intosaid cells a DNA sequence encoding said one or more proteins, peptides,polypeptides or analogs in the form of a suitable vector carrying saidsequence, said vector being capable of effecting the insertion of saidsequence into said cells in a way that said sequence is expressed insaid cells.
 21. A method for the modulation of the TNF or FAS-R ligandeffect on cells mediated by p55 TNF-R and FAS-R, or the functionsmediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by otherproteins containing a ‘death domain’ motif, comprising: (a) constructinga recombinant animal virus vector carrying a sequence encoding a viralsurface protein (ligand) that is capable of binding to a specific cellsurface receptor on the surface of said cell to be treated and a secondsequence encoding a protein selected from the proteins, peptides,polypeptides and analogs according to claim 14, said protein, peptide,polypeptide or analogs, when expressed in said cells being capable ofmodulating the activity of said ‘death domain’ motif-containing protein,and (b) infecting said cells with said vector of (a).
 22. A method formodulating the TNF or FAS-R ligand effect on cells mediated by p55 TNF-Rand FAS-R, or the functions mediated in cells by NGF-R, MORT-1, RIP,TRADD, ankyrin 1 or by other proteins containing a ‘death domain’ motif,comprising treating said cells with antibodies, or active fragments orderivatives thereof, specific for a protein, peptide, polypeptide, oranalog in accordance with claim 14, said treating being by applicationof a suitable composition containing said antibodies, active fragmentsor derivatives thereof to said cells, said composition being formulatedfor intracellular application.
 23. A method for modulating the TNF orFAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or thefunctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1 orby other proteins containing a ‘death domain’ motif, comprising treatingsaid cells with an oligonucleotide sequence encoding an antisensesequence of at least part of the sequence according to claim 9, saidoligonucleotide sequence being capable of blocking the expression of atleast one of the ‘death domain’ motif-binding proteins or peptides. 24.A method according to claim 23, wherein said treating of said cells isby transfection of said cells with a recombinant animal virus vectorcomprising the steps of: (a) constructing a recombinant animal virusvector carrying a sequence encoding a viral surface protein (ligand)that is capable of binding to a specific cell surface receptor on thesurface of said cell to be treated and in a second sequence encodingsaid oligonucleotide sequence; and (b) infecting said cells with saidvector of (a).
 25. A method for treating tumor cells or HIV-infectedcells or other diseased cells, comprising: (a) constructing arecombinant animal virus vector carrying a sequence encoding a viralsurface protein that is capable of binding to a specific tumor cellsurface receptor or HIV-infected cell surface receptor or receptorcarried by other diseased cells and a sequence encoding a proteinselected from the group consisting of the proteins, peptides,polypeptides and analogs of claim 14, said protein, peptide, polypeptideor analog when expressed in said tumor, HIV-infected, or other diseasedcell, being capable of killing said cell; and (b) infecting said tumoror HIV-infected cells or other diseased cells with said vector of (a).26. A method for modulating the TNF or FAS-R ligand effect on cellsmediated by p55 TNF-R and FAS-R, or the functions mediated in cells byNGF-R, MORT-1, RIP, TRADD, ankyrin 1 or by other proteins containing a‘death domain’ motif, comprising applying the ribozyme procedure inwhich a vector encoding a ribozyme sequence capable of interacting witha cellular mRNA sequence encoding a protein or peptide according toclaim 14, is introduced into said cells in a form that permitsexpression of said ribozyme sequence in said cells, and wherein whensaid ribozyme sequence is expressed in said cells it interacts with saidcellular mRNA sequence and cleaves said mRNA sequence resulting in theinhibition of expression of said protein or peptide in said cells.
 27. Apharmaceutical composition for the modulation of the TNF or FAS-Rligand-effect on cells mediated by p55 TNF-R and FAS-R, or the functionsmediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1, or by otherproteins containing a ‘death domain’ motif, comprising, as activeingredient, a modulator according to claim
 1. 28. A pharmaceuticalcomposition for modulating the TNF or FAS-R ligand effect on cellsmediated by p55 TNF-R and FAS-R, or the functions mediated in cells byNGF-R, MORT-1, RIP, TRADD, ankyrin 1, or by other proteins containing a‘death domain’ motif, comprising, as active ingredient, a recombinantanimal virus vector encoding a protein capable of binding a cell surfacereceptor and encoding a protein or peptide or analogs thereof accordingto claim
 14. 29. A pharmaceutical composition for modulating the TNF orFAS-R ligand effect on cells mediated by p55 TNF-R and FAS-R, or thefunctions mediated in cells by NGF-R, MORT-1, RIP, TRADD, ankyrin 1, orby other proteins containing a ‘death domain’ motif, comprising, asactive ingredient, an oligonucleotide sequence encoding an antisensesequence of the sequence according to claim
 9. 30. A method forisolating and identifying a protein capable of binding to the ‘deathdomain’ motifs of ‘death domain’ motif-containing proteins, comprisingapplying the procedure of non-stringent Southern hybridization followedby PCT cloning, in which a sequence, or parts thereof, according toclaim 9 is used as a probe to bind sequences from a cDNA or genomic DNAlibrary, having a least partial homology thereto, said bound sequencesthen being amplified and cloned by the PCR procedure to yield clonesencoding proteins having at least partial homology to said sequences ofclaim
 9. 31. A method for modulating the concerted intracellularmodulation resulting from the indirect interaction between the FAS-R andthe p55 TNF-R, via their cascading interaction of MORT-1 and TRADD, orMORT-1 and RIP, comprising, treating cells with a modulator which willenhance or inhibit the MORT-1-TRADD or MORT-1-RIP mediated p55TNF-R-FAS-R interaction.